Controlled oxidation of hydrocarbons by the membrane-bound methane monooxygenase: the case for a tricopper cluster

[Figure: see text]. The growing need for inexpensive methods to convert methane to methanol has sparked considerable interest in methods that catalyze this process. The integral membrane protein particulate methane monooxygenase (pMMO) mediates the facile conversion of methane to methanol in methanotrophic bacteria. Most evidence indicates that pMMO is a multicopper enzyme, and these copper ions support redox, dioxygen, and oxo-transfer chemistry. However, the exact identity of the copper species that mediates the oxo-transfer chemistry remains an area of intense debate. This highly complex enzyme is notoriously difficult to purify because of its instability outside the lipid bilayer and tendency to lose its essential metal cofactors. For this reason, pMMO has resisted both initial identification and subsequent isolation and purification for biochemical and biophysical characterization. In this Account, we describe evidence that pMMO is a multicopper protein. Its unique trinuclear copper cluster mediates dioxygen chemistry and O-atom transfer during alkane hydroxylation. Although a recent crystal structure did not show this tricopper cluster, we provide compelling evidence for such a cluster through redox potentiometry and EPR experiments on the "holo" enzyme in pMMO-enriched membranes. We also identify a site in the structure of pMMO that could accommodate this cluster. A hydrophobic pocket capable of harboring pentane, the enzyme's largest known substrate, lies adjacent to this site. In addition, we have designed and synthesized model tricopper clusters to provide further chemical evidence that a tricopper cluster mediates the enzyme's oxo-transfer chemistry. These biomimetic models exhibit similar spectroscopic properties and chemical reactivity to the putative tricopper cluster in pMMO. Based on computational analysis using density functional theory (DFT), triangular tricopper clusters are capable of harnessing a "singlet oxene" upon activation by dioxygen. An oxygen atom is then inserted via a concerted process into the C-H bond of an alkane in the transition state during hydroxylation. The turnover frequency and kinetic isotope effect predicted by DFT show excellent agreement with experimental data.     

Determination of the carbon kinetic isotope effects on propane hydroxylation mediated by the methane monooxygenases from Methylococcus capsulatus (Bath) by using stable carbon isotopic analysis

Authentic propane with known position-specific carbon isotope composition at each carbon atom was subjected to hydroxylation by the particulate and soluble methane monooxygenase (pMMO and sMMO) from Methylococcus capsulatus (Bath), and the corresponding position-specific carbon isotope content was redetermined for the product 2-propanol. Neither the reaction mediated by pMMO nor that with sMMO showed an intermolecular (12)C/(13)C kinetic isotope effect effect on the propane hydroxylation at the secondary carbon; this indicates that there is little structural change at the carbon center attacked during formation of the transition state in the rate-determining step. This finding is in line with the concerted mechanism proposed for pMMO (Bath), and suggested for sMMO (Bath), namely, direct side-on insertion of an active "O" species across the C-H bond, as has been previously reported for singlet carbene insertion.     

Crucial Role of the Chaperonin GroES/EL for Heterologous Production of the Soluble Methane Monooxygenase from Methylomonas methanica MC09

Methane is a widespread energy source and can serve as an attractive C1 building block for a future bioeconomy. The soluble methane monooxygenase (sMMO) is able to break the strong C−H bond of methane and convert it to methanol. The high structural complexity, multiplex cofactors, and unfamiliar folding or maturation procedures of sMMO have hampered the heterologous production and thus biotechnological applications. Here, we demonstrate the heterologous production of active sMMO from the marine Methylomonas methanica MC09 in Escherichia coli by co-synthesizing the GroES/EL chaperonin. Iron determination, electron paramagnetic resonance spectroscopy, and native gel immunoblots revealed the incorporation of the non-heme diiron centre and homodimer formation of active sMMO. The production of recombinant sMMO will enable the expansion of the possibilities of detailed studies, allowing for a variety of novel biotechnological applications.     

Structural and mechanistic insights into methane oxidation by particulate methane monooxygenase

Particulate methane monooxygense (pMMO) is an integral membrane copper-containing enzyme that converts methane to methanol. Knowledge of how pMMO selectively oxidizes methane under ambient conditions could impact the development of new catalysts. The crystal structure of Methylococcus capsulatus (Bath) pMMO reveals the composition and location of three metal centers. Spectroscopic data provide insight into the coordination environments and oxidation states of these metal centers. These results, combined with computational studies and comparisons to relevant systems, are discussed in the context of identifying the most likely site for O 2 activation.     

Dioxygen activation in soluble methane monooxygenase

The controlled oxidation of methane to methanol is a chemical transformation of great value, particularly in the pursuit of alternative fuels, but the reaction remains underutilized industrially because of inefficient and costly synthetic procedures. In contrast, methane monooxygenase enzymes (MMOs) from methanotrophic bacteria achieve this chemistry efficiently under ambient conditions. In this Account, we discuss the first observable step in the oxidation of methane at the carboxylate-bridged diiron active site of the soluble MMO (sMMO), namely, the reductive activation of atmospheric O(2). The results provide benchmarks against which the dioxygen activation mechanisms of other bacterial multicomponent monooxygenases can be measured. Molecular oxygen reacts rapidly with the reduced diiron(II) cen-ter of the hydroxylase component of sMMO (MMOH). The first spectroscopically characterized intermediate that results from this process is a peroxodiiron(III) species, P*, in which the iron atoms have identical environments. P* converts to a second peroxodiiron(III) unit, H(peroxo), in a process accompanied by the transfer of a proton, probably with the assistance of a residue near the active site. Proton-promoted O-O bond scission and rearrangement of the diiron core then leads to a diiron(IV) unit, termed Q, that is directly responsible for the oxidation of methane to methanol. In one section of this Account, we provide a detailed discussion of these processes, with particular emphasis on possible structures of the intermediates. The geometries of P* and H(peroxo) are currently unknown, and recent synthetic modeling chemistry has highlighted the need for further structural characterization of Q, currently assigned as a di(μ-oxo)diiron(IV) "diamond core." In another section of the Account, we discuss in detail proton transfer during the O(2) activation events. The role of protons in promoting O-O bond cleavage, thereby initiating the conversion of H(peroxo) to Q, was previously a controversial topic. Recent studies of the mechanism, covering a range of pH values and in D(2)O instead of H(2)O, confirmed conclusively that the transfer of protons, possibly at or near the active site, is necessary for both P*-to-H(peroxo) and H(peroxo)-to-Q conversions. Specific mechanistic insights into these processes are provided. In the final section of the Account, we present our view of experiments that need to be done to further define crucial aspects of sMMO chemistry. Here our goal is to detail the challenges that we and others face in this research, particularly with respect to some long-standing questions about the system, as well as approaches that might be used to solve them.     

Catalytic and Spectroscopic Properties of the Halotolerant Soluble Methane Monooxygenase Reductase from Methylomonas methanica MC09

The soluble methane monooxygenase receives electrons from NADH via its reductase MmoC for oxidation of methane, which is itself an attractive C1 building block for a future bioeconomy. Herein, we present biochemical and spectroscopic insights into the reductase from the marine methanotroph Methylomonas methanica MC09. The presence of a flavin adenine dinucleotide (FAD) and [2Fe2S] cluster as its prosthetic group were revealed by reconstitution experiments, iron determination and electron paramagnetic resonance spectroscopy. As a true halotolerant enzyme, MmoC still showed 50 % of its specific activity at 2 M NaCl. We show that MmoC produces only trace amounts of superoxide, but mainly hydrogen peroxide during uncoupled turnover reactions. The characterization of a highly active reductase is an important step for future biotechnological applications of a halotolerant sMMO.     

Evaluation of methane oxidation in rice plant-soil system

Mechanisms of methane oxidation in the plant-soil system of rice were studied in a pot experiment using two cultivars (PSBRc-30 and IR72) at two growth stages (flowering and heading). Methane emission was measured by chambers, while methane oxidation was determined through propylene amendment as an alternative substrate to be propylene oxide (PPO) and acetylene as an inhibitor for methane oxidizing (methanotrophic) bacteria. Cell numbers (methanotrophic and methanogenic bacteria) were determined by the most probable number method. The cultivar PSBRc-30 consistently showed higher methane emission rates than IR72. Methane flux clearly decreased from flowering to heading stages in both cultivars. This observation was largely reflected by trends in the mechanisms involved: either methanogenic cell numbers or activities decreased with plant age while methanotrophic cell numbers or activities generally showed an increasing trend. The methanogenic population was in the order of 10⁵ g^–1 dry soil, while the population of methanotrophs ranged from 10⁴ to nearly 10⁶ g^–1 dry soil. Methanotrophic activity followed the order; root (1.7–2.8 nL PPO g^–1 DM h^–1) > shoot (0.7–2.0) > soil (0–0.4) when the consumption of alternative substrate was related to dry matter. Derived from the estimated amounts of soil and plant biomass in the pot experiment, however, the soil generally accounted for more than 90% of the total methane oxidation. Within the plant segments, methane oxidation activities in the root exceeded those of the shoot by factor of approximately 10.     

Methane oxidation in soils with different textures and land use

Intact core samples from soils with different textures and land use were tested for their capacity to oxidise methane. The soil cores were taken from arable land, grassland and forest. It was found that coarse textured soils (6.74–16.38 μg CH₄ m^-2 h^-1) showed a higher methane uptake rate than fine textured soils (4.66–5.34 μg CH₄ m^-2 h^-1). Increasing soil tortuosity was thought to reduce the methane oxidation rate in fine textured soils. The oxidation rate of forest soils (16.32–16.38 μg CH₄ m^-2 h^-1), even with a pH below 4.5, was very pronounced and higher than arable land (11.40–14.47 μg CH₄ m^-2 h^-1) and grassland (6.74–9.30 μg CH₄ m^-2 h^-1). Within the same textural class arable land showed a faster methane uptake rate than grassland. In grassland with a fine texture, even methane production was observed. Nitrogen availability and turnover in these land use systems were thought to cause the different oxidation rates. Decreasing the moisture content slowed down the oxidation rate in all soils. This could be caused by an increased N turnover and a starvation of the methanotrophic bacteria. This revised version was published online in August 2006 with corrections to the Cover Date.     

Biomimetic oxidation studies. 7. Alkane functionalization with a MMO structural model, [Fe2O(OAc)(tris((1-methylimidazol-2-yl) methyl) amine)2]3+, in the presence oft-butyl hydroperoxide and oxygen gas

A biomimetic structural model of the active site of methane monooxygenase enzyme, [Fe₂O(OAc)(tris((1-methylimidazol)-2-methyl)amine₂]³⁺, 1, has been shown to functionalize cyclohexane, toluene, adamantane, propane, and ethane in the presence oft-butyl hydroperoxide and oxygen gas. A mechanism is proposed to account for these results which implicates an alkyl hydroperoxide intermediate to the alcohol, ketone, and aldehyde products in an oxygen gas dependent reaction, while aldehyde and ketone products can also be formed from the further oxidation of the alcohols in an oxygen gas independent reaction.Partially presented at the 12th North American Catalysis Society Meeting, Lexington, KY, May 1991, Abstract D 26 and the 8th ISHC meeting in August 2–7, 1992, Amsterdam, The Netherlands, Abstract O–13. For previous biomimetic oxidation papers see ref. [1 ].     

Homogeneous, catalytic, oxidative coupling of methane to acetic acid in one step

Acetic acid is an important petrochemical that is currently produced from methane (or coal) in a three-step process based on carbonylation of methanol. We report a direct, selective, oxidative condensation of two methane molecules to acetic acid at 180°C in concentrated sulfuric acid. ¹³C isotopic labeling studies show that both carbons of acetic acid originate from two methane molecules in a formal eight-electron redox reaction. The reaction is catalyzed by palladium and the results are consistent with the reaction occurring by tandem catalysis involving methane C–H activation to generate Pd-CH₃ species, followed by efficient oxidative carbonylation with methanol, generated in situ from methane, to produce acetic acid. Control Experiments suggest that the carbonylation occurs via the in situ oxidation of CH₃OH to low levels of CO.     

Preparation and effect of Mo-V-Cr-Bi-Si oxide catalysts on controlled oxidation of methane to methanol and formaldehyde

Mo-Cr-V-Bi-Si multi-component oxide catalysts were synthesized by three different coprecipitation methods and used in the controlled oxidation of methane to methanol and formaldehyde. It was shown that Mo content in Mo-V-Cr-Bi-Si oxides and the performance of these catalysts were strongly influenced by different coprecipitation methods. The highest methanol and formaldehyde selectivity of 80.2% could be achieved at a methane conversion of 10 % for the catalyst prepared by a particular method. The results of XRD indicated that the crystalline phase structures of catalysts were sensitive to Mo, V and Bi loadings. Bi(III) could combine with V(V) and Mo(VI) to form BiVO₄ and γ-Bi₂MoO₆, whereas Cr seemed to form a single Cr₂O₂ crystalline phase in the presence of Bi. The effects of Mo and Cr loading on controlled methane oxidation were also investigated. Mo(VI) oxide appears to favor the formation of partial oxidation products and Cr(III) oxide seems to enhance the conversion of methane.     

A novel cyclic process for synthesis gas production

This paper reports a novel cyclic partial oxidation process for the production of synthesis gas with the in situ separation of oxygen from air. A perovskite-type oxide, La_0.8Sr_0.2Co_0.5Fe_0.5O_3−δ, is employed in the experimental study of the process as an oxygen retaining material. Air and methane are periodically brought into contact with the oxide packed in a fixed-bed reactor. The oxide, during the air step, exclusively retains oxygen while partial oxidation reaction occurs during the methane step utilizing the retained oxygen. Although combustion and cracking reactions occur in addition to the partial oxidation reaction during the methane step, high methane conversion is achieved and hydrogen and CO are produced with high selectivity.     

Partial oxidation of methane with air for methanol production in a post-plasma catalytic system

Partial oxidation of methane to methanol via post-plasma catalysis using a dielectric-barrier discharge was performed under mild reaction conditions. Air was used as the oxidizing co-reactant because of its economical practicality. Three catalysts impregnated with Pt, Fe₂O₃, CeO₂ on ceramic supports located downstream of the discharge zone were examined for increased selectivity towards methanol. It was found that all three catalysts had no significant effect on the conversion of methane, but enhanced methanol selectivity, which could be explained by a two-stage reaction mechanism. The Fe₂O₃-based catalyst showed the best catalytic activity, and high stability in the reaction. The methanol selectivity of the Fe₂O₃-assisted plasma process was 36% higher than that of the non-catalytic system at a rather low catalyst temperature (150 °C). In addition, the effects of input power, discharge frequency, discharge gap distance, total flow rate, and methane/air ratio on methane conversion and methanol yield were also studied.     

Methane Oxidation in Pig and Cattle Slurry Storages, and Effects of Surface Crust Moisture and Methane Availability

Storages with liquid manure (slurry) may develop a surface crust of particulate organic matter, or an artificial crust can be established. Slurry storages are net sources of atmospheric methane (CH₄), but a potential for bacterial oxidation of CH₄ in surface crusts was recently suggested in a study of experimental storages. The present study was conducted to investigate methanotrophic activity under practical storage conditions. Surface crusts from slurry storages at two pig farms and four dairy farms were sampled in late autumn. Mixed samples (0–4 cm depth) were used to determine changes in CH₄, O₂ and CO₂ during incubation, while intact subsamples were used to characterize CH₄ oxidation as a function of CH₄ availability and moisture content. Methane oxidation was observed in all materials except for an expanded clay product (Leca) sampled from a pig slurry storage. Despite significant variation between replicate subsamples, there was a significant increase in methanotrophic activity when CH₄ concentrations increased from 500 to 50,000 ppmv. Maximum fluxes ranged from −1 to −4.5 g CH₄ m⁻² d⁻¹. Surface crust samples were partly dried and then re-wetted in four steps to the original moisture content, each time followed by determination of CH₄ fluxes. Only one surface crust material showed a relationship between CH₄ fluxes and moisture content that would implicate gas diffusivity in the regulation of CH₄ oxidation. The occurrence of inducible CH₄ oxidation activity in slurry storage surface crusts indicates that there is a potential for stimulating the process by manipulation of gas phase composition above the stored slurry.     

Carbon dioxide reforming of methane with a free energy minimization approach

Carbon dioxide reforming of methane to syngas is one of the primary technologies of the new poly-generation energy system on the basis of gasification gas and coke oven gas. A free energy minimization is applied to study the influence of operating parameters (temperature, pressure and methane-to-carbon dioxide ratio) on methane conversion, products distribution, and energy coupling between methane oxidation and carbon dioxide reforming methane. The results show that the methane conversion increases with temperature and decreases with pressure. When the methane-to-carbon dioxide ratio increases, the methane conversion drops but the H₂/CO ratio increases. By the introduction of oxygen, an energy balance in the process of the carbon dioxide reforming methane and oxidation can be realized, and the CO/H₂ ratio can be adjusted as well without water-gas shift reaction for Fischer-Tropsch or methanol synthesis. This work was presented at the 6 ^th Korea-China Workshop on Clean Energy Technology held at Busan, Korea, July 4–7, 2006.     

Synthesis of well-dispersed PtRuSnOx by ultrasonic-assisted chemical reduction and its property for methanol electrooxidation

PtRuSnO_x supported on multi-wall carbon nanotubes (MWCNTs) was prepared by ultrasonic-assisted chemical reduction method. The as-prepared catalyst was characterized by X-ray diffraction (XRD) and transmission electron microscopy (TEM). The XRD patterns indicate that Pt exists as the face-centered cubic structure, Ru is alloyed with platinum, while non-noble metal oxide SnO_x exists as an amorphous state. From TEM observation, PtRuSnO_x is well dispersed on the surface of MWCNTs with the particle size of several nanometers. The electrochemical properties of the as-prepared catalyst for methanol electrooxidation were studied by cyclic voltammetry (CV) and chronoamperometry (CA). The onset potential of methanol oxidation on PtRuSnO_x and PtRu catalysts is much more negative than that on Pt catalyst, shifting negatively by about 0.20 V, while the peak current density of methanol oxidation on PtRuSnO_x is higher than that on PtRu. Electrochemical impedance spectroscopy (EIS) studies also show that the reaction kinetics of methanol oxidation is improved with the presence of SnO_x. The addition of non-noble metal oxide SnO_x to PtRu promotes the catalytic activity for methanol electrooxidation and the possible reaction mechanism is proposed.     

Exfoliated graphene-supported Pt and Pt-based alloys as electrocatalysts for direct methanol fuel cells

To greatly improve the electrocatalytic activity for methanol oxidation, high-quality exfoliated graphene decorated with uniform Pt nanocrystals (NCs) (3 nm) have been prepared by a very simple, low-cost and environmentally benign process. During the entire process, no surfactant and no halide ions were involved, which not only enabled very clean surface of Pt/graphene leading to excellent conductivity, but also greatly improved the electrocatalyst tolerance to carbon monoxide poisoning (Pt/graphene, I_f/I_b = 1.197), compared to commercial Pt/C (I_f/I_b = 0.893) catalysts. To maximize the electrocatalytic performance and minimize the amount of precious Pt, Pt–M/graphene (M = Pd, Co) hybrids have also been prepared, and these hybrids have much larger electrochemically active surface areas (ECSA), which are 4 (PtPd/graphene) and 3.3 (PtCo/graphene) times those of commercial Pt/C. The PtPd/graphene and PtCo/graphene hybrids also have remarkably increased activity toward methanol oxidation (I_f/I_b = 1.218 and 1.558). Furthermore, density functional theory (DFT) simulations demonstrate that an electronic interaction occurred between Pt atoms and graphene, indicating that graphene substrate plays a crucial role in regulating the electron structure of attached Pt atom, which confirmed that the increased efficiency of methanol oxidation was due to the synergetic effects of the hybrid structure.     

Specular reflectivity studies of the adsorption and the oxidation of methanol on palladium in an alkaline solution

Adsorption and anodic oxidation of methanol on palladium electrode in 0·5 N NaOH was studied by measuring current—potential (i-E) and reflectivity—potential (R/R₀-E) curves. Presence of methanol retarded the formation of the oxide layer of Pd, giving rise to an anodic shift of the potential. A linear relation was found between the potential retarded and the logarithm of methanol concentration, the slope being about 30 mV/decade. The form of adsorbed entities was discussed based on the slope. The cathodic charge consumed by the reduction of Pd-oxide was smaller in the presence of methanol than in the absence. This was explained by three causes; the less amount of oxide layer formed during the anodic sweep owing to the adsorption of oxidation product of methanol, the reduction of formed oxide layer by a chemical reaction with methanol, and a compensation effect due to the anodic oxidation of methanol during the cathodic sweep. Plottings of the total charge in the anodic region vs — ΔR/R₀ in the absence and the presence of methanol suggested that electro-oxidation of methanol proceeds only at the bare portion of Pd.     

On the role of WO3 surface hydroxyl groups for the photocatalytic partial oxidation of methane to methanol

The photocatalytic partial oxidation of methane to methanol has been investigated on WO₃. The surface fluorination of the catalyst has given an insight on the reaction mechanism which proceeds mainly through the interaction with surface hydroxyl groups.     

Effects of methanol co-feeding in F–T synthesis on a silica supported Co-catalyst

The effects of methanol on the performance of Co/SiO₂ catalyst for Fischer–Tropsch synthesis were studied in a fixed-bed reactor at 210 °C and 2.0 MPa. It was found that external addition of methanol brought about an increase in the selectivity of methane, water and CO₂ and a decrease in the activity of the catalyst. FTIR and TPSR characterizations proved that methanol first converted to the methoxy groups and water and then the methoxy groups reacted with hydrogen to produce methane, which led to the increase of the selectivity of methane and water. And the increase of the selectivity of CO₂ was ascribed to the water–gas shift reaction. The decline of the activity might result from the instantaneous oxidation of the metallic cobalt due to the sudden addition of methanol and the formation of cobalt silicates and/or hydrosilicates species. More methanol introduction into the feeding gas would result in the formation of larger amount of silicates species, which led to the more serious deactivation of the catalyst. In addition, the hydrogen was found necessary for the chain propagation whereas CO was not. The methanol might substitute for CO to perform the chain initiation.